Antibodies Research Articles

Structure and sequence engineering approaches to improve invivo expression of nucleic acid-delivered antibodies.

Monoclonal antibodies are an important class of biologics with over 160 Food and Drug Administration/European Union-approved drugs. A significant bottleneck to global accessibility of recombinant monoclonal antibodies stems from complexities related to their production, storage, and distribution. Recently, gene-encoded approaches such as mRNA, DNA, or viral delivery have gained popularity, but ensuring biologically relevant levels of antibody expression in the host remains a critical issue. Using a synthetic DNA platform, we investigated the role of antibody structure and sequence toward invivo expression. SARS-CoV-2 antibody 2196 was recently engineered as a DNA-encoded monoclonal antibody (DMAb-2196). Utilizing an immunoglobulin heavy and light chain "chain-swap" methodology, we interrogated features of DMAb-2196 that can modulate invivo expression through rational design and structural modeling. Comparing these results to natural variation of antibody sequences resulted in development of an antibody frequency score that aids in the prediction of expression-improving mutations by leveraging antibody repertoire datasets. We demonstrate that a single amino acid mutation identified through this score increases invivo expression up to 2-fold and that combinations of mutations can also enhance expression. This analysis has led to a generalized pipeline that can unlock the potential for invivo delivery of therapeutic antibodies across many indications.

Rethinking Optimal Immunogens to Face SARS-CoV-2 Evolution Through Vaccination.

SARS-CoV-2, which originated in China in late 2019, quickly fueled the global COVID-19 pandemic, profoundly impacting health and the economy worldwide. A series of vaccines, mostly based on the full SARS-CoV-2 Spike protein, were rapidly developed, showing excellent humoral and cellular responses and high efficacy against both symptomatic infection and severe disease. However, viral evolution and the waning humoral neutralizing responses strongly challenged vaccine long term effectiveness, mainly against symptomatic infection, making necessary a strategy of repeated and updated booster shots. In this repeated vaccination context, antibody repertoire diversification was evidenced, although immune imprinting after booster doses or reinfection was also demonstrated and identified as a major determinant of immunological responses to repeated antigen exposures. Considering that a small domain of the SARS-CoV-2 Spike protein, the receptor binding domain (RBD), is the major target of neutralizing antibodies and concentrates most viral mutations, the following text aims to provide insights into the ongoing debate over the best strategies for vaccine boosters. We address the relevance of developing new booster vaccines that target the evolving RBD, thus focusing on the relevant antigenic sites of the SARS-CoV-2 new variants. A combination of this strategy with immunofusing and computerized approaches could minimize immune imprinting, therefore optimizing neutralizing immune responses and booster vaccine efficacy.

The antibody repertoire of autoimmune sensory neuronopathies targets pathways of the innate and adaptative immune system. An autoantigenomic approach.

The antibody repertoire of autoimmune sensory neuronopathies targets pathways of the innate and adaptative immune system. An autoantigenomic approach.

Expression Cloning of Antibodies from Single Human B Cells.

The majority of lymphomas originate from B cells at the germinal center stage. Preferential selection of B-cell clones by a limited set of antigens has been suggested to drive lymphoma development. While recent studies in B-cell chronic lymphocytic leukemia (CLL) have shown that self-reactive B-cell receptors (BCR) can generate cell-autonomous signaling and proliferation, our knowledge about the role of BCRs for the development or survival of other lymphomas remains limited. Here, we describe a strategy to characterize the antibody reactivity of human B cells. The approach allows for unbiased characterization of the human antibody repertoire on single-cell level through the generation of recombinant monoclonal antibodies from primary human B cells of defined origin. This protocol offers a detailed description of the method starting from the flow cytometric isolation of single human B cells to the RT-PCR-based amplification of the expressed immunoglobulin (Ig) transcripts (IGH, IGK, and IGL) and their subsequent cloning into expression vectors for the in vitro production of recombinant monoclonal antibodies. The strategy may be used to obtain information about the clonal evolution of B-cell lymphomas by single-cell sequencing of Ig transcripts and on the antibody reactivity of human lymphoma B cells.

Immunoglobulins: Structure, Function, and Therapeutic Applications in Immune Response

The article titled "Immunoglobulins and Its Applications" provides a comprehensive overview of the structure, function, and therapeutic significance of immunoglobulins, also known as antibodies. These vital proteins, produced by plasma cells in response to immunogens, are essential components of the immune system, playing a critical role in recognizing and neutralizing pathogens such as bacteria and viruses. The article elaborates on the five primary classes of immunoglobulins (IgA, IgD, IgE, IgG, and IgM), each distinguished by its unique structural and functional properties. It discusses the molecular composition of immunoglobulins, highlighting the significance of their variable and constant regions in antigen binding. The paper also explores the diverse immune functions mediated by these proteins, including precipitation, agglutination, and neutralization of antigens. Additionally, the article emphasizes the therapeutic applications of immunoglobulins, particularly in treating immune deficiencies and other diseases. Advances in monoclonal antibody technology have significantly expanded the utility of immunoglobulins in both research and medicine, leading to the development of innovative therapeutic approaches such as humanized monoclonal antibodies and bifunctional antibodies. This comprehensive analysis underscores the critical role of immunoglobulins in both natural immunity and therapeutic interventions

A method for screening functional anti-Treg antibodies using a Treg-like cell line.

Regulatory T cells can suppress activated T cell proliferation by direct cell-contact, although the exact mechanism is poorly understood. Identification of a Treg-specific cell surface molecule that mediates suppression would offer a unique target for cancer immunotherapy to inhibit Treg immunosuppressive function or deplete Tregs in the tumor microenvironment. In this study, we explored a method of whole cell immunization using a Treg-like cell line (MoT cells) to generate and screen monoclonal antibodies that bound cell surface proteins in their native conformations and functionally reversed Treg-mediated suppression. From the 105 hybridomas that bound to the MoT cell surface, a functional screen utilizing conventional Treg suppression assays revealed 32 candidate antibodies that exhibited functional activity (reversed or enhanced suppressive activity). As an example, we characterized one anti-MoT mAb, 12E7, that exhibited strong binding to MoT cells and conventional Treg cell surfaces. This candidate antibody was subsequently found to bind to a potential suppressive target, CD44, and demonstrated the ability to partially reverse MoT and conventional human Treg-mediated suppression.

Rho(D) immune globulin shortage and fetal Rh(D) screening with cell-free DNA.

Despite the availability of Rh(D) immune globulin (RhIg) to prevent alloimmunization in Rh(D)-negative pregnant patients, anti-Rh(D) alloimmunization remains a prevalent cause of hemolytic disease of the fetus and newborn (HDFN). Recent RhIg shortages have caused clinicians and professional societies to identify methods to prioritize RhIg administration. New cell-free DNA (cfDNA) tests to predict fetal red blood cell antigen genotypes have been proposed as an option to prioritize the administration of RhIg to Rh(D)-negative pregnant people. Commercial laboratories offer fetal Rh(D) genotype testing as part of cfDNA screening for fetal aneuploidy. Studies indicate that these tests have a high sensitivity and specificity for the detection of fetal Rh(D) status. Considering the current RhIg shortage, the American College of Obstetricians & Gynecologists (ACOG) suggests that utilizing cfDNA tests to determine fetal Rh(D) status is a reasonable approach to prioritize RhIg administration when supply is limited. cfDNA screening for fetal Rh(D) status is a reasonable approach to triage the administration of RhIg in the setting of the current RhIg shortage. Utilization of cfDNA screening for fetal Rh(D) and other red blood cell antigen status is likely to increase in routine care. Research, professional society guidance, and education are necessary to ensure well tolerated and equitable utilization.

Direct detection of 4-dimensions of SARS-CoV-2: infection (vRNA), infectivity (antigen), binding antibody, and functional neutralizing antibody in saliva

We developed a 4-parameter clinical assay using Electric Field Induced Release and Measurement (EFIRM) technology to simultaneously assess SARS-CoV-2 RNA (vRNA), nucleocapsid antigen, host binding (BAb) and neutralizing antibody (NAb) levels from a drop of saliva with performance that equals or surpasses current EUA-approved tests. The vRNA and antigen assays achieved lower limit of detection (LOD) of 100 copies/reaction and 3.5 TCID₅₀/mL, respectively. The vRNA assay differentiated between acutely infected (n = 10) and infection-naïve patients (n = 33) with an AUC of 0.9818, sensitivity of 90%, and specificity of 100%. The antigen assay similarly differentiated these patient populations with an AUC of 1.000. The BAb assay detected BAbs with an LOD of 39 pg/mL and distinguished acutely infected (n = 35), vaccinated with prior infection (n = 13), and vaccinated infection-naïve patients (n = 13) from pre-pandemic (n = 81) with AUC of 0.9481, 1.000, and 0.9962, respectively. The NAb assay detected NAbs with a LOD of 31.6 Unit/mL and differentiated between COVID-19 recovered or vaccinated patients (n = 31) and pre-pandemic controls (n = 60) with an AUC 0.923, sensitivity of 87.10%, and specificity of 86.67%. Our combo assay represents a significant technological advancement to simultaneously address SARS-CoV-2 infection and immunity, and it lays the foundation for tackling potential future pandemics.

Klebsiella pneumoniae Glycoconjugate Vaccine Leads Based on Semi-Synthetic O1 and O2ac Antigens.

Klebsiella pneumoniae (KP) is a common opportunistic pathogen that emerged as a new critical threat to human health, due to its hypervirulence and widespread resistance against many antibiotics, including carbapenems. Alternative intervention strategies such as vaccines are not available. Cell-surface lipopolysaccharides (LPS) and capsular polysaccharides (CPS) are attractive targets for vaccine development. We present a method to synthesize LPS substructures, covering over 70 % of virulent KP strains that were used to characterize the antibody repertoire of infected patients. Thereby, glycoconjugate vaccine leads against the Klebsiella pneumoniae serotypes O1, O2a, O2afg and O2ac have been identified.

Klebsiella pneumoniae Glycoconjugate Vaccine Leads Based on Semi‐Synthetic O1 and O2ac Antigens

Klebsiella pneumoniae (KP) is a common opportunistic pathogen that emerged as a new critical threat to human health, due to its hypervirulence and widespread resistance against many antibiotics, including carbapenems. Alternative intervention strategies such as vaccines are not available. Cell‐surface lipopolysaccharides (LPS) and capsular polysaccharides (CPS) are attractive targets for vaccine development. We present a method to synthesize LPS substructures, covering over 70% of virulent KP strains that were used to characterize the antibody repertoire of infected patients. Thereby, glycoconjugate vaccine leads against the Klebsiella pneumoniae serotypes O1, O2a, O2afg and O2ac have been identified.

Distinct Neutralising and Complement-Fixing Antibody Responses Can Be Induced to the Same Antigen in Haemodialysis Patients After Immunisation with Different Vaccine Platforms.

Background/Objectives: Generalised immune dysfunction in chronic kidney disease, especially in patients requiring haemodialysis (HD), significantly enhances the risk of severe infections. Vaccine-induced immunity is typically reduced in HD populations. The SARS-CoV-2 pandemic provided an opportunity to examine the magnitude and functionality of antibody responses in HD patients to a previously unencountered antigen-Spike (S)-glycoprotein-after vaccination with different vaccine platforms (viral vector (VV); mRNA (mRV)). Methods: We compared the total and functional anti-S antibody responses (cross-variant neutralisation and complement binding) in 187 HD patients and 43 healthy controls 21-28 days after serial immunisation. Results: After 2 doses of the same vaccine, HD patients had anti-S antibody levels and a complement binding capacity comparable to controls. However, 2 doses of mRV induced greater polyfunctional antibody responses than VV (defined by the presence of both complement binding and cross-variant neutralisation activity). Interestingly, an mRV boost after 2 doses of VV significantly enhanced antibody functionality in HD patients without a prior history of SARS-CoV-2 infection. Conclusions: HD patients can generate near-normal, functional antigen-specific antibody responses following serial vaccination to a novel antigen. Encouragingly, exploiting immunological memory by using mRNA vaccines and boosting may improve the success of vaccination strategies in this vulnerable patient population.

Transcriptome of capsular contracture around breast implants mimics allograft rejection: a matched case-control study.

Capsular contracture is a frequent and severe complication following breast implant surgery. Although several theories on the pathophysiology exist, the exact molecular mechanisms remain unclear. This study aimed to identify the specific genes, signaling pathways, and immune cells associated with capsular contracture. Breast implant capsule biopsies were collected from women undergoing implant replacement after breast augmentation. Patients with capsular contracture (Baker III/IV) and healthy controls (Baker I) were included in equal numbers and matched based on implant brand, surface, plane, and rupture status. Whole transcriptome RNA-sequencing was used for gene expression profiling. We analyzed biopsies from 51 breasts of 50 women, revealing 1,500 differentially expressed genes based on capsular contracture status. Our findings revealed that capsular contracture signaling pathways mimic allograft rejection with activation of both the innate immune system (e.g., IL1A/B, CXCl9, TREML4, CR1) and the adaptive immune system (e.g., CD80 and IFN-γ). Capsular contracture was associated with increased expression of macrophages, CD4+ T cells, B cells, and plasma cells with upregulation of several immunoglobulin genes (e.g., IGHD/IGHE). Moreover, several fibrosis-related genes were significantly upregulated (e.g., MMP1, MMP1, MMP12) and downregulated (TIMP4) in breasts with capsular contracture. The results indicate that B cells play a more crucial role in the development of capsular contracture than previously assumed. The disease mechanism resembles allograft rejection, indicating that capsular contracture is a form of immunological rejection of the breast implant. This study identified key genes associated with capsular contracture, suggesting new drug candidates e.g., MMP1 inhibitors to improve breast implant surgery outcomes. Synergizing research on allograft rejection and capsular contracture could also lead to new treatment strategies.

The vital role of biological standardization in ensuring efficacy and safety of biological products - Historical perspectives.

Biological Standardization has been pivotal to the success of traditional biological products, such as vaccines, antitoxins, and immune globulins, by ensuring their quality and consistency across manufacturers worldwide. The principles of biological standardization have similarly supported the development and manufacture of safe and effective modern biological products, including hormone, therapeutic protein, and monoclonal antibody products, and continue to play a vital role in advancing new cutting-edge biological products, such as tissue, cellular, and gene-therapy products. Biological standardization started with the physical standards ensuring the reliability and suitability of methods used to test biological products and science of bioassays or biological methods and related biostatistics providing a framework for evaluating biological, functional activity or potency of these products. It expanded to include written standards defining the quality requirements for manufacturing and regulation of biological standards. Due to the shift in the biologics industry from public health to commercial-driven enterprises during the past 50 years, the biological standardization program has evolved to include the product-specific reference standards and harmonization of physical standards. The global success of conventional vaccines in controlling numerous deadly infectious diseases can largely be attributed to the availability of physical and written international standards developed through a strong biological standardization program. This article explores the evolution of biological standardization for more than a century, its scientific and regulatory principles, challenges from disruption in international standardization efforts, and future perspectives for the field.

Morbilidad de la infección por el SARS-CoV-2 en adultos jóvenes en la altura en un centro de salud militar

Introduction: In the face of this debutant disease generated by SARS-CoV-2 the clinical and epidemiological characteristics are new in each region, city or hospital environment in which patients with this infection occur. Objective: Determine the morbidity of SARS-CoV-2 infection in young adults living at high altitudes. Method: Descriptive, quantitative, observational, retrospective, cross-sectional study, carried out at 3,259 meters above sea level, identified the clinical-epidemiological characteristics of patients with a positive result for SARS-CoV-2 with immunochromatography, a sample of 97 patients hospitalized at the Military Health Center No. 31 Chilca-Huancayo of the 31st Infantry Brigade in the period from May to June 2020. Results: A total of 97 male patients (100%) were studied, with a mean age of 21,5 ± 3. The employment status was: active (49,5%), reservist (50,5%). Grade: soldier (25.8%), corporal (23,7%), sergeant 1st. (14,4%), 2nd Sergeant (35,1%), non-commissioned officer (1,0%). The rapidtest was positive (100.0%), IgM–IgG immunoglobulin positive (96.9%), IgG positive (3,1%). Respiratory rate: normal (96,9%), tachypnea (3,1%). Heart rate: normal (63,9%), tachycardia (32,0%), bradycardia (4,1%). Temperature: normal (89,7%), low-grade fever (10,3%). Oxygensaturation: mild hypoxia (56,7%), normal (40,2%), moderate hypoxia (3,1%). Conclusions: The epidemiological profile of the study are young adults, reservists predominate, Sergeant 2nd.; symptomatic with mild hypoxia and diarrhea; oligosymptomatic in respiratory rate, heart rate, body temperature, sore throat, general discomfort, nausea, vomiting, headache, skin rash; and asymptomatic in cough, nasal congestion, smell and consciousness disturbances.

Molecular measurable residual disease by immunoglobulin gene rearrangements on circulating tumor DNA predicts outcome in diffuse large B-cell lymphoma.

In diffuse large B-cell lymphoma (DLBCL) treatment response relies on imaging. We investigated the potential value of molecular measurable residual disease (MRD) on circulating tumor DNA (ctDNA) to predict patient outcomes. We retrospectively evaluated 73 patients. Analyses were conducted on 57 tumor biopsies, based on sample availability. At baseline, next-generation sequencing was used to detect clonal immunoglobulin (IG) gene rearrangements on tumor biopsies and ctDNA. MRD monitoring was applied by tracking the IG clones in ctDNA samples collected during treatment (interim) and at the end of treatment (EOT). MRD results were correlated with clinical data and radiologic disease assessment. Before treatment, clonal IG were found in 91.2% (52/57) of tumor biopsies and in 93.2% (68/73) of ctDNA samples. In paired samples, the same clonotype was found in 69.2% (36/52) of cases. At the interim analysis, ctDNA MRD was negative in 32/45 evaluable patients and positive in 13/45, correlating significantly with progression-free survival (PFS) (78.1% MRD- vs 30.8% MRD+; p.

Induction of Fc-dependent functional antibodies against different variants of SARS-CoV-2 varies by vaccine type and prior infection

BackgroundSARS-CoV-2 transmission and COVID-19 disease severity is influenced by immunity from natural infection and/or vaccination. Population-level immunity is complicated by the emergence of viral variants. Antibody Fc-dependent effector functions are as important mediators in immunity. However, their induction in populations with diverse infection and/or vaccination histories and against variants remains poorly defined.MethodsWe evaluated Fc-dependent functional antibodies following vaccination with two widely used vaccines, AstraZeneca (AZ) and Sinovac (SV), including antibody binding of Fcγ-receptors and complement-fixation in vaccinated Brazilian adults (n = 222), some of who were previously infected with SARS-CoV-2, as well as adults with natural infection only (n = 200). IgG, IgM, IgA, and IgG subclasses were also quantified.ResultsAZ induces greater Fcγ-receptor-binding (types I, IIa, and IIIa/b) antibodies than SV or natural infection. Previously infected individuals have significantly greater vaccine-induced responses compared to naïve counterparts. Fcγ-receptor-binding is highest among AZ vaccinated individuals with a prior infection, for all receptor types, and substantial complement-fixing activity is only seen among this group. SV induces higher IgM than AZ, but this does not drive better complement-fixing activity. Some SV responses are associated with subject age, whereas AZ responses are not. Importantly, functional antibody responses are well retained against the Omicron BA.1 S protein, being best retained for Fcγ-receptor-1 binding, and are higher for AZ than SV.ConclusionsHybrid immunity, from combined natural exposure and vaccination, generates strong Fc-mediated antibody functions which may contribute to immunity against evolving SARS-CoV-2 variants. Understanding determinants of Fc-mediated functions may enable future vaccines with greater efficacy against different variants.

The physiological landscape and specificity of antibody repertoires are consolidated by multiple immunizations.

Diverse antibody repertoires spanning multiple lymphoid organs (i.e., bone marrow, spleen, lymph nodes) form the foundation of protective humoral immunity. Changes in their composition across lymphoid organs are a consequence of B-cell selection and migration events leading to a highly dynamic and unique physiological landscape of antibody repertoires upon antigenic challenge (e.g., vaccination). However, to what extent B cells encoding identical or similar antibody sequences (clones) are distributed across multiple lymphoid organs and how this is shaped by the strength of a humoral response remains largely unexplored. Here, we performed an in-depth systems analysis of antibody repertoires across multiple distinct lymphoid organs of immunized mice and discovered that organ-specific antibody repertoire features (i.e., germline V-gene usage and clonal expansion profiles) equilibrated upon a strong humoral response (multiple immunizations and high serum titers). This resulted in a surprisingly high degree of repertoire consolidation, characterized by highly connected and overlapping B-cell clones across multiple lymphoid organs. Finally, we revealed distinct physiological axes indicating clonal migrations and showed that antibody repertoire consolidation directly correlated with antigen specificity. Our study uncovered how a strong humoral response resulted in a more uniform but redundant physiological landscape of antibody repertoires, indicating that increases in antibody serum titers were a result of synergistic contributions from antigen-specific B-cell clones distributed across multiple lymphoid organs. Our findings provide valuable insights for the assessment and design of vaccine strategies.

Semisynthetic Glycoconjugate Vaccine Lead against Klebsiella pneumoniae Serotype O2afg Induces Functional Antibodies and Reduces the Burden of Acute Pneumonia.

Carbapenem-resistant Klebsiella pneumoniae (CR-Kp) bacteria are a serious global health concern due to their drug-resistance to nearly all available antibiotics, fast spread, and high mortality rate. O2afg is a major CR-Kp serotype in the sequence type 258 group (KPST258) that is weakly immunogenic in humans. Here, we describe the creation and evaluation of semisynthetic O2afg glycoconjugate vaccine leads containing one and two repeating units of the polysaccharide epitope that covers the surface of the bacteria conjugated to the carrier protein CRM197. The semisynthetic glycoconjugate containing two repeating units induced functional IgG antibodies in rabbits with opsonophagocytic killing activity and enhanced complement activation and complement-mediated killing of CR-Kp. Passive immunization reduced the burden of acute pneumonia in mice and may represent an alternative to antimicrobial therapy. The semisynthetic glycoconjugate vaccine lead against CR-Kp expressing O2afg antigen is awaiting preclinical development.

Factors affecting the diagnostic delay of myasthenia gravis.

Early detection and diagnosis of myasthenia gravis (MG) is important to improve the chance of remission and overall prognosis. This study aims to investigate the factors affecting the diagnostic delay of MG thereby highlighting the challenges in the diagnostic process. We conducted a retrospective study examining characteristics and factors involved in the diagnostic process of MG. We included 350 patients treated for MG at the Copenhagen Neuromuscular Unit (CNMC), between 1980 and 2022. A total of 315 patients had data on time to diagnosis, which was used to categorize them into an early-and a delayed diagnosis group, based on a median cut-off of 141 days. A separate analysis was conducted for the cases with an extremely delayed time to diagnosis exceeding the upper quartile range of 864 days (n = 28). Finally, change in time to MG diagnosis over time was identified. The overall mean time to diagnosis was found to be 331days (range: 5-4492days). Patients who experienced a delayed diagnosis were significantly younger at onset (50.4 vs. 55.4years, p-value < 0.05), and experienced higher frequency of asymptomatic periods (29% vs. 10%, p-value < 0.05). We found a delayed referral to specialists, extended time spent at specialists, and delayed initiation of paraclinical diagnostic tests. Additionally, in the delayed group we found extended time from the initial antibody (AB) evaluation to the first positive result (3.2 vs. 86.1 days, p-value < 0.05). Individuals in the early diagnosis group may be referred to stroke specialist earlier, however, this finding was not statistically significant (18 vs. 464days, p-value 0.17). Gender, presenting symptom, final type of MG and socioeconomic status where not associated with delayed diagnosis. Generally, a trend of decreasing time to diagnosis over time was observed. Younger age at onset, higher prevalence of asymptomatic periods, and a delayed positive AB titer test may be risk factors for a delayed MG diagnosis. Prompt referral to a stroke specialist may lead to an earlier diagnosis.

Experimental and Computational Studies on Domain-Swapped Structure Stabilization of an Antibody Light Chain by Disulfide Bond Introduction.

Development of different platforms would be useful for designing functional antibodies to improve the efficiency of antibody-based drugs. Three-dimensional domain swapping (3D-DS) may occur in the variable region of antibody light chain #4C214A, and a pair of domain-swapped dimers may interact with each other to form a tetramer. In this study, to stabilize the 3D-DS dimer structure in #4C214A, Val2 in strand A (swapping region) and Thr97 in strand G were replaced with Cys residues, generating #4 V2C/T97C/C214A with a Cys2-Cys97 disulfide bond that cross-links strands A and G of different protomers. The #4 V2C/T97C/C214A tetramer did not dissociate into monomers at low protein concentration (6 μM); however, some of the tetramers were converted to monomers by disulfide bond reduction. Two-dimensional free energy profile analysis for the tetramerization of two 3D-DS dimers was performed by molecular dynamics simulation. These results show that disulfide bond introduction is useful for controlling the dimerization/dissociation of the variable region through 3D-DS.